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Sphingomonas sp. strain Fr1 has recently been shown to protectArabidopsis thaliana against the bacterial leaf pathogenPseudomonas syringae DC3000. Here, we describe a forward geneticin planta screen to identify genes inSphingomonas sp. Fr1 necessary for this effect. About 5,000Sphingomonas sp. Fr1 mini-Tn5 mutants were assayed for a defect in plant protection against aluxCDABE -taggedP. syringae DC3000 derivative in a space-saving 24-well plate system. The bioluminescence of the pathogen was used as the indicator of pathogen proliferation and allowed for the identification ofSphingomonas sp. Fr1 mutants that had lost the ability to restrict pathogen growth before disease symptoms were visible. Potential candidates were validated using the same miniaturized experimental system. Of these mutants, 10 were confirmed as plant protection defective yet colonization competent. The mutants were subsequently evaluated in a previously described standard microbox system, and plants showed enhanced disease phenotypes after pathogen infection relative to those inoculated with the parental strain as a control. However, the disease severities were lower than those observed for control plants that were grown axenically prior to pathogen challenge, which suggests that several traits may contribute to plant protection. Transposon insertion sites of validated mutants with defects in plant protection were determined and mapped to 7 distinct genomic regions. In conclusion, the established screening protocol allowed us to identify mutations that affect plant protection, and it opens the possibility to uncover traits important forin planta microbe-microbe interactions.

applied and environmental microbiology

Forward Genetic <i>In Planta</i> Screen for Identification of Plant-Protective Traits of Sphingomonas sp. Strain Fr1 against Pseudomonas syringae DC3000

Forward Genetic In Planta Screen for Identification of Plant-Protective Traits of Sphingomonas sp. Strain Fr1 against Pseudomonas syringae DC3000